Tissue specific promoters

ABSTRACT

The invention relates to tissue specific promoters which can be used in plants for one or more of the following purposes: a. altering carbohydrate metabolism b. preventing memory substance loss c. expression of an invertase inhibitor d. expression of a fructosyl transferase e. expression of a levan sucrase f. expression of genes coding for transported proteins for N-compounds g. expression of characteristics which increase resistance/tolerance with respect to pathogens.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to promoters and their use in transgeneticplants.

It is possible to genetically alter crop plants by molecular biologymethods, and to express proteins specifically. For this, the selectionof a suitable promoter is of considerable relevance. Therefore, a greatneed for well-characterized promoters with specific characteristicsexists.

2. Description of the Related Art

During the last years, a large number of plant promoters was isolatedand analyzed for their effect. In the meantime, octopine synthase (ocs),nopaline synthase (nos) and mannopine synthase (mas) isolated fromAgrobacterium tumefaciens and TR promoters (De Greve et al., 1982,Depicker et al., 1982; Velten et al., 1984) and 35S promoter ofcauliflower mosaic virus (Odell et al., 1985) respectively, have found abroad application. Plant promoters with a constitutive activity havebeen described for tobacco (WO 97/28268) and raspberry (WO 97/27307).

Organ, tissue or cell specific promoters can be used for the expressionof genes in specific plant parts. Specificity in this context can mean,that a promoter is mainly or exclusively active in one organ, tissue orcell type. Mainly active in a particular organ are, e.g. the tomatopromoters TFM7 and TFM9 in tomato fruits (U.S. Pat. No. 5,608,150), abrassica promoter in roots (WO 94/02619), a sun flower promoter in seeds(WO 98/45460) and a potato promoter (WO 98/18940) in leaves. Thesepromoters show their highest activity in the mentioned organs. Anexclusive activity for a certain compartment was described for a guardcell specific promoter of potato (DE 42 07358 A1) for the tapetumspecific promoter TA29 from tobacco (EP 0 344 029 B1) and for the pistiland pollen specific SLG promoter from brassica (Dzelzkàlns et al.,1993).

From sugar beet, an organ specific promoter is known, which is mainlyactive in the storage root tissue of sugar beet (WO 97/32027). However,this promoter of the sucrose synthase gene is not only active in rootsbut also, with less activity, in other tissues like leaves (Hesse andWillmitzer, 1996).

SUMMARY OF THE INVENTION

Therefore, it is the problem of the present invention to provide newpromoters and plants with the possibility of tissue specific expressionof genes either in roots or in above-ground plant parts.

This problem is solved according to the invention by promoters accordingto the main claim and a transgenetic plant obtainable by transformationof a plant cell with a promoter according to the main claim, which isoperatively linked to a transferred gene, and subsequent regeneration ofthe transgenetic plant.

Some of the terms used in this application are specified below:

The term promoter refers to a nucleotide sequence, which regulates theexpression of a gene under its control, if necessary in dependency ofendogenous and exogenous factors. Among these factors are, e.g.inductors, repressors and similar DNA binding proteins as well asenvironmental influences. A promoter may consist of several elements.However, it at least comprises one regulatory element, which isresponsible for the transcription of the gene under its control.

A promoter, which is active in above-ground and chloroplast containingplant parts, as leaves, and not in below-ground organs, shows adetectable activity in leaves, measured by RNA blots, which, undercomparable experimental conditions, is detectible in below-ground organsof the plant to less than 20%, preferably to less than 10% and morepreferably to less than 5%. This specificity is not restricted to aparticular experimental time point, but is generally present during theentire vegetation period.

A promoter, which is active in below-ground organs and not in aboveorgans of the plant, for example shows in roots at detectable activitymeasured by RNA blots, which is, under comparable experimentalconditions, detectable in above-ground organs of the plant likepetioles, leaves and blossoms to less than 20%, preferably to less than10% and more preferably to less than 5%. This specificity is notrestricted to a particular experimental time point, but is generallypresent during the entire vegetation period.

“Derivatives” of a promoter are shortened or elongated or sectionwiseidentical versions of this promoter or homologes with the same, modifiedor singular characteristics.

“Inducible by pathogens” means the action of external factors on theplant, which result in a defence reaction of the plant. These can beattacks of insects (bites), bacteria, fungi, nematodes or otherpathogens, but also abiotic influences as mechanical wounding (e.g. byhail-storm).

“Direct antifungal activity” means that gene products act directlyantifungal by, e.g. dissolving cell walls or by coding for phytoalexinesynthases and metabolites, respectively, which inhibit the fungalmetabolism.

“Indirect antifungal activity” means, that gene products activate theplant gene defense. Among these genes are, e.g. resistant genes,components of signal transduction (as kinases, phosphatases),transcription factors or enzymes, which produce signal substances (asenzymes forming ethylene, salicylic acid or jasmonate, enzymes formingreactive oxygen species, enzymes forming nitrogen monoxide).

“Sink leaves” are such leaves, which, due to their small size, consumemore carbohydrates than they produce themselves.

“Source leaves” are leaves, which, due to their size, produce morecarbohydrate then they consume themselves.

By “infection” is meant the earliest time point, at which the metabolismof the fungus (the growth of the fungus) is prepared for a penetrationof the host tissue. Among these are, e.g. the growth of hypha or theformation of specific infection structures, as penetration hypha andappressoria.

The expression “homology” hereby means a homology of at least 70% on DNAbasis, which can be determined according to known methods, e.g. computerassisted sequence comparisons (S. F. Altschul et al. (1990), Basic LocalAlignment search tool, J. Mol. Biol. 215: 403-410).

Complementary nucleotide sequence means with respect to adouble-stranded DNA that the second DNA strand, which is complementaryto the first DNA strand, comprises the nucleotide bases, whichcorrespond to the bases of the first strand according to the rules forbase pairing.

The term “hybridizing” as it is used herein means hybridizing underconventional conditions, as they are described in Sambrook et al.(Molecular Cloning. A laboratory manual, Cold Spring Harbor LaboratoryPress, 2^(nd) edition, 1989), preferably under stringent conditions.Stringent hybridization conditions are for example: hybridizing in 4×SSCat 65° C. and subsequent multiple washing in 0.1×SSC at 65° C. for atotal of 1 hour. Less stringent hybridization conditions are forexample: hybridizing in 4×SSC at 37° and subsequent multiple washing in1×SSC at room temperature. The term “stringent hybridization conditions”as used herein can also mean: hybridizing at 68° C. in 0.25 M sodiumphosphate, pH 7.2, 7% SDS, 1 mM EDTA and 1% BSA for 16 hours andsubsequent washing twice with 2×SSC and 0.1% SDS at 68° C.

In the following, the invention is described in more detail, referringto the figures and examples.

The promoters and their derivatives according to the invention areparticularly characterized by the fact that they are exclusively activein roots or in above-ground organs of a plant. With their help,transgenetic plants with particular characteristics can be produced. Ina preferred manner, they can be used for the following purposes:

-   a. amendment of the carbohydrate metabolism-   b. avoidance of storage substances losses-   c. expression of an invertase inhibitor-   d. expression of a fructosyl transferase-   e. expression of a levan sucrase-   f. expression of genes coding for transporter proteins for    N-compounds-   g. development of features, which increase the resistance/tolerance    towards pathogens.

The promoters according to SEQ ID NO: 1 and SEQ ID NO:2 are active inroots, in particular of sugar beet, but not in above-ground organs ofthis plant. This characteristic can be used for the improvement of themetabolism of transgenetic plants, in particular the carbohydratemetabolism of sugar beets. It is an improvement of the carbohydratemetabolism to reduce the loss of sucrose and the accumulation of glucoseand fructose during the storage of beet bodies after harvest. The use ofan invertase inhibitor gene under the expression control of SEQ ID NO: 1and SEQ ID NO: 2 can reduce the activity of vacuolar invertase in theroot. By organ specific expression of the inhibitor, pleitropic effectsare avoided, which inhibit the invertase in the entire plant.

Further improvements of the carbohydrate metabolism are the productionof the sweetener palatinite or the synthesis of polyfructanes in theroot of sugar beets under the use of the described sequences.

The root specific active promoters (SEQ ID NO: 1 and SEQ ID NO: 2) canalso be used to improve the nitrogen metabolism of the plants. For this,transport protein genes for ammonium (NH₄ ⁺), nitrate (NO₃ ⁻) andnitrite (NO₂ ⁻) ions are over-expressed in the root, and the uptake ofthe mentioned ions is increased. A further improvement of theN-metabolism is the reduced incorporation of “bad nitrogen” in thestorage organs of the plant. Elevated concentrations of N-compounds instorage organs often reduce the nutrition physiological value of harvestproducts or hamper the isolation of storage substances, as sucrose, fromsugar beet roots. A reduced incorporation of “bad nitrogen” in the rootcan be achieved by a reduced uptake of ammonium and nitrate ions fromthe soil. For this purpose root specific active promoters are used, inorder to reduce the expression of endogenous transporter genes organspecifically by, e.g. an “anti-sense” approach.

The promoters according to the invention can also be used to improve theresistance to diseases of the plants.

Viral infections of the sugar beet are often restricted to only oneorgan as the root or the above-ground plant parts. Thus, the virus BNYVVinfects and colonizes primarily the beet root, and yellowing virusesBMYV and BYV are found only in leaves. Root active promoters andpromoters, which are only active in above-ground organs can be used toto obtain organ specificity via the virus resistance concepts, which arebased on gene silencing and anti-sense technique, respectively.

Sequences and Figures

The nucleotide sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 andSEQ ID NO: 4 are depicted in 5′-3′-orientation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 and 2 show exclusively root specific expression of gene 2-1-48and 2-1-36 during root development by a RNA blot experiment. 10 μg totalcell RNA, which had been isolated from different organs of 4, 6, 10, 12,16 and 22 week old sugar beets, respectively, was separated in adenaturated formaldehyde agarose gel. RNA was isolated from the taproot,the lateral roots, sink and source leaves and, in case of 4 week oldplants, from the hypocotyl and the seed leaves. The cDNA fragment 2-1-48and 2-1-36, respectively, was used as hybridization probe.

FIG. 3 shows by an RNA blot experiment, that gene 2-3-9 is expressedduring the beet development exclusively in above-ground plant organs. 10μg total cell RNA, which had been isolated from different organs of 4,6, 10, 12, 16 and 22 week old sugar beets, respectively, were separatedin a denaturated formaldehyde agarose gel. RNA was isolated from thetaproot, the lateral roots, sink and source leaves and, in the case of 4week old plants, from the hypocotyl and the seed leaves. The cDNAfragment 2-3-9 was used as hybridization probe.

FIG. 4 shows in a DNA blot experiment that in the genome of sugar beetgenotypes 1K0088 two copies of the gene 2-3-9 and in genotype 4B5421only one gene copy are/is present. 10 μg genomic DNA was used forrestriction digest, respectively. The cDNA fragment 2-3-9 was used ashybridization probe.

On the basis of the restriction analyzes, which have been carried out,FIG. 5 shows the location and orientation of the coding region of thefirst copy of gene 2-3-9 and of the C1 promoter for 5 isolated lambdaphages. Further, subcloning of the insert of phage λ 6.1.1 into plasmidspc1a and pc1b is depicted. Hatched box indicates the coding region forthe first copy of gene 2-3-9.

FIG. 6 shows the 5.19 kb reporter gene vector pluc-nos2. The plasmidpluc-nos2 comprises the luciferase gene from Photinus pyralis and thenos-terminator. The multiple cloning site in the 5′-region of thereporter gene allows the insertion of promoter fragments. Therestriction enzymes in brackets cut the plasmid several times.

FIG. 7 shows the 6.34 kb reporter gene construct pc1L-1097. The vectorpc1L-1097 is formed by insertion of the C1 promoter fragment (position1-1145 of SEQ ID NO: 3) into vector pluc-nos2. The restriction enzymesin brackets cut the plasmid several times.

FIG. 8 shows the 12.44 kb reporter gene construct pc1L-7126. Afterisolation of the 6029 bp genomic 5′-region of copy 1 of gene 2-3-9 fromvector pc1 b, the DNA fragment was inserted into vector pc1L-1097. Theresulting vector pc1L-7126 comprises the regulatory 5′-region of thefirst copy of gene 2-3-9 from position 1-7126. The restriction enzymesin brackets cut the plasmid several times.

FIG. 9 shows the 8.1 kb reporter gene construct pc2L-2998. The vectorpc2L-2998 is formed by insertion of the C2 promoter fragment (position1-3046 of the nucleotide sequence of SEQ ID NO: 4) into vectorpluc-nos2. The restriction enzymes in brackets cut the plasmid severaltimes.

FIG. 10 shows the 6.9 kb reporter gene construct pc2L-1827. The vectorpc2L-1827 is formed by a 5′-deletion of the C2 promoter of plasmidpc2L-2998. The C2 promoter in vector pc2L-1827 comprises the nucleotidepositions 1172-3046 of nucleotide sequence SEQ ID NO: 4. The restrictionenzymes in brackets cut the plasmid several times.

FIG. 11 shows the 6.04 kb reporter gene construct pc2L-989. The vectorpc2L-989 is formed by a 5′-deletion of the C2 promoter of plasmidpc2L-2998. The C2 promoter in vector pc2L-989 comprises the nucleotidepositions 2011-3046 of nucleotide sequence SEQ ID NO: 4. The restrictionenzymes in brackets cut the plasmid several times.

FIG. 12 shows the 5.39 kb reporter gene construct pc2L-342. The vectorpc2L-342 is formed by a 5′-deletion of the C2 promoter of plasmidpc2L-2998. The C2 promoter in vector pc2L-342 comprises the nucleotidepositions 2657-3046 of nucleotide sequence SEQ ID NO: 4. The restrictionenzymes in brackets cut the plasmid several times.

FIG. 13 shows the activity of reporter gene constructs pc2L-2998,pc2L-1827, pc2L-989 and pc2L-342 after ballistic transformation in sugarbeet leaves. Per construct 1-2 DNA preparations, each with 4experimental repeats were used. The calculated Photinus pyralisluciferase activities were normalized by parallel measurement of Renillareniformis luciferase activity, and variations in the transformationefficiency were thereby compensated.

FIG. 14 shows the 15.07 kb binary plant transformation vector pc1G-1097.The C1 promoter is translationally fused with the gus reporter gene. TheC1 promoter comprises the nucleotide positions 1-1145 of the nucleotidesequence of SEQ ID NO:3.

FIG. 15 shows the 17 kb binary plant transformation vector pc2G-2998.The C2 promoter is translationally fused with the gus reporter gene. TheC2 promoter comprises the nucleotide positions 1-3046 of the nucleotidesequence of SEQ ID NO: 4.

FIG. 16 shows the histochemical detection of the activity of the C1promoter in leaves of transgenetic brassica plants. Leaf pieces of thetransformant pc1G-1097-86 (right) are colored in blue due to gusreporter gene activity, in comparison to the non-transgenetic control(left).

FIG. 17 shows the histochemical detection of the activity of the C1promoter in leaves of transgenetic tobacco plants. A leaf piece of thetransformant pc1G-1097-3 (right) is colored in blue due to gus reportergene activity, in comparison to the non-transgenetic control (left).

FIG. 18 shows a DNA sequence comparison between the conserved regions ofthe C1 and the C2 promoter. In the figure, the positions of GATA boxes,I box, GT-1 binding sites, CAAT box, two circadian boxes and TATA boxesare indicated. The translational start of the first copy of gene 2-3-9is at position 1098, and the translational start of the second copy ofgene 2-3-9 is at position 2998. The transcription initiation pointwithin the C1 promoter is at position 984, and the transcriptioninitiation point within the C2 promoter is at position 2928.

EXAMPLES Isolation of Root and Leaf Specific Expressed cDNAs of SugarBeet

According to the method of suppression subtractive hybridization(Diatchenko et al., 1996) an accumulation of cDNA fragments of genesexpressed in leaves and the taproot of sugar beet was conducted. Forthis, from both tissues first total RNA and then polyA(+) RNA wasisolated. Additionally, total RNAs from sprouts and inflorescences wereisolated, which were used for Northern blot analysis.

The following experiments were carried out according to the protocol ofthe PCR select systems of the company CLONTECH. With polyA(+) RNA fromleaf and taproot, cDNA was synthesized. With both cDNAs two subtractionswere carried out. For the accumulation of leaf specific genes, the leafcDNA as a probe was subtracted against the cDNA from taproot. For theaccumulation of taproot specific genes, the cDNA from taproot as a probewas subtracted against the cDNA from leaf. The subtraction was carriedout precisely according to the protocol of the CLONTECH kit, and alsothe internal control was carried out. All further molecular biologyexperiments were carried out according to standard protocols (Sambrooket al., 1989). After subtraction, amplified cDNA fragments wereobtained, which were either accumulated with leaf or root specificgenes.

For further analysis, the cDNA fragments were cloned into the TA cloningvector pCR2.1 (invitrogen) and transformed into E. coli. A blue-whiteselection allowed the identification of recombinant plasmids (Sambrooket al., 1989). In white colonies, the expression of β-galactosidase issuppressed by an insert, which leads to white colonies, because theenzyme substrate, which is added to the medium is no longer cleaved. Perμg of PCR product, approximately 300 white colonies were obtained. Intotal, 62 clones of the subtraction of taproot specific genes and 60clones of the subtraction of leaf specific genes were furthercharacterized. For this, the DNA of the clones was cleaved with therestriction enzyme RsaI. This isolated the inserts from adaptors. Then,half of the cleaved DNA was electrophoretically separated on gels, andthe DNA was transferred to nylon membranes. For the identification ofDNA fragments, which were specifically accumulated, the filters with thePCR products (cDNA fragments) were hybridized with both subtractions.cDNA fragments, which hybridized with the cDNA fragments from which theywere subcloned but not or not as strong with the cDNA fragments from theother subtraction were further analyzed, because they representedpotentially accumulated or tissue specific genes.

Among 62 clones from the subtraction of taproot specific genes, 18 wereidentified, which hybridized strongly with the cDNA fragments of thesubtraction of taproot specific genes but not or only weakly with thecDNA fragments of the subtraction of leaf specific genes. Among 60clones from the subtraction of leaf specific genes, 23 were identified,which hybridized strongly with the cDNA fragments of the subtraction ofleaf specific genes but not or only weakly with the cDNA fragments ofthe subtraction of taproot specific genes. All identified cDNA insertsof both subtractions were entirely sequenced, and compared to each otherwith a sequence analysis program (Pileup, GCG Wisconsin AnalysisPackage). Based on similarities (homologous) of the sequences among eachother, in total 9 different cDNA fragments of the subtraction of taprootspecific genes and 14 different cDNA fragments of the subtraction ofleaf specific genes remained.

All 9 different cDNA fragments of the subtraction of taproot specificgenes and 7 of 14 different cDNA fragments of the subtraction of leafspecific genes were hybridized by Northern blot analysis with RNA fromleaf, taproot, sprout and inflorescence. Three clones, 2-1-36, 2-1-48and 2-3-9 showed a very specific hybridization pattern. 2-1-36 and2-1-48 hybridized only with RNA from taproot and not with RNA from leaf,sprout and inflorescence, and 2-3-9 hybridized only with RNA from greentissue and not with RNA from taproot.

Genes 2-1-36 and 2-1-48 are Expressed Exclusively in the Sugar Beet RootDuring the Vegetation Period

In order to analyze the expression behavior of genes 2-1-36 and 2-1-48during the entire vegetation period, sugar beet seeds are applied to thefield. During the central European vegetation period 5, complete sugarbeet plants are harvested after 4, 6, 10, 12, 16 and 22 week,respectively after sowings. The plants show at no time signs fordiseases. Total cell RNA is isolated according to Logemann et al., 1987from the organs sink and source leaf, petiole, lateral root and taproot(root body). The expression of the genes is determined by RNA blotanalyses.

For the analyses of development dependent gene expression with an RNAblot, 10 μg total cell RNA per organ and time point are separated in adenaturated formaldehyde agarose gel, as described in Sambrook et al.,(1989). Electrophoretically separated RNA is transferred by capillarblot technique (Sambrook et al., 1989) onto a hybond N nylon membrane(Amersham Pharmacia Biotech, Freiburg). Radioactive labeling of 20 ng of2-1-36 and 2-1-48 cDNA fragments with 50 μCi ³²P-dATP (6000 Ci/mMol,Amersham Pharmacia Biotech, Freiburg), respectively, is carried out withthe help of Prime-It II random primer kit (Stratagene GmbH, Heidelberg)according to the manufacturer's instructions. The subsequenthybridization of the RNA filter with labeled probe is carried out in 20ml hybridization buffer (50% formamide, 5×SSC, 5× Denhard's, 1% SDS, 0.1mg herring sperm DNA, 40 mM sodium phosphate puffer pH 6.8) at 42° C. ina hybridization oven (Biometra GmbH, Goettingen) according to Sambrooket al., 1989. After hybridization, the nylon membrane is washed andexposed on an X-ray film (Kodak BioMax MS, Kodak AG, Stuttgart) in thepresence of an intensifying screen (Kodak BioMax MS, IntensifyingScreen, Kodak AG, Stuttgart) for 6-24 h at −80° C. The development ofthe X-ray film is then done in X-ray film developer and X-ray film fixer(Tetenal Photowerk GmbH and Co., Norderstedt).

The RNA blot hybridized with probe 2-1-48 shows that the gene 2-1-48 in4 week old sugar beet is only expressed in the root and in the rootedhead (hypocotyl) and in 6, 10, 12, 16 and 22 week old sugar beetexclusively in the taproot and the lateral roots (FIG. 1). At no timepoint an expression of gene 2-1-48 can be observed in the above-groundplant organs petiole, source and sink leaf.

The RNA blot was evaluated with the help of a phosphoimager (BioimagingAnalyzer BAS 1000, Fujiy Japan), in order to quantify the transcriptaccumulation. The data of the quantification are depicted in table 1.

The accumulation of a transcript, which is detectable by probe 2-1-48and thus a corresponding promoter activity is strongly expressed intaproots of 4, 6 and 10 week old plants. In 12 week old roots, the geneexpression reaches its maximum and then decreases significantly in 16and 22 week old plants. Therefore, the gene 2-1-48 is strongly and verystrongly expressed in root bodies of young beets and plants of middleage, respectively, and significantly decreases with increasing age. Inthe above-ground plant organs it is only possible to detected a veryweak gene expression in petioles and leaves in young beets. Thedetectable transcript amount in 6, 10 and 12 week old petioles is only3, 2 and 1.2%, respectively of the transcript amount detectable in thetaproot at these time points. The detected transcript amount in 6 and 10week old sink and source leaves is 2.5% and 1.6 and 2.1%, respectivelyof the accumulated 2-1-48 transcripts in the taproot at these timepoints.

The use of the cDNA clone 2-1-36 as hybridizing probe for thedevelopment specific RNA blot shows that the gene is expressed in 4 weekold sugar beets only in the root and the rooted head (hypoctoyl) and in6, 10, 12, 16 and 22 week old plants exclusively in the taproot and inthe lateral roots (FIG. 2). In the above-ground organs petiole, sink andsource leaf, a transcript is at no time point visible. The expression ofgene 2-1-36 is not constitutive in the root and the lateral roots butdevelopment dependent. This tendency is already visible on theautoradiogram of the RNA blot, but is even more pronounced after thequantification of the labeled filter by the phosphoimager (table 2).While gene 2-1-36 is expressed during the juvenile development of thebeet in 4, 6 and 10 week old taproots and 6 and 10 week old lateralroots weakly but increasingly stronger, beets and lateral roots show avery strong increase of expression after 12 weeks. The expression ofgene 2-1-36 decreases slightly in taproots at the time points 16 and 22weeks in comparison to the 12 week value, but it remains on a highlevel. In lateral roots of 16 week old plants, there is also a decreaseof 2-1-36 expression in comparison to the 12 weeks value. However, theexpression of 2-1-36 in this organ increases in 22 week old plants againto the high 12 week level. Therefore, the gene 2-1-36 is expressedweakly in 4-10 week old plants and is expressed strongly and thereforedevelopment dependent in 12-22 week old plants. In the above-groundorgans sink and source leaf and petiole, a significant transcript amountcannot be quantified at any time point. All measured values in theseorgans are in the range of background.

Gene 2-3-9 is Exclusively Expression in Above-Ground Plant Organs DuringBeet Development

In order to analyze the expression of gene 2-3-9 during the entirevegetation period, the RNA blots for the expression analysis of rootspecific expressed genes were hybridized with probe 2-3-9. These RNAblots were prepared using RNA of the organs sink and source leaf,petiole, taproot and lateral root of 4, 6, 10, 12, 16 and 22 week oldsugar beets, as described.

The hybridization result of the RNA blot shows, as depicted in FIG. 3,that gene 2-3-9 is expressed at any analysis time point in sink andsource leaves and petioles. An expression in the below-ground plantorgans lateral and taproot is at no time point optically visible.Quantification of the RNA blot with the help of a phosphoimager confirmsthe optical impression and shows the absence of 2-3-9 transcripts intaproots and lateral roots and a strong transcript accumulation inleaves and petioles (table 3). The measured values for the taproot andlateral root are calculated to zero and are in the region of variationof the background. Therefore, gene 2-3-9 is exclusively expressed inabove-ground plant parts during the entire vegetation period.

Gene 2-3-9 is Present in Different Copy Numbers in Different Sugar BeetGenotypes

The number of genomic copies of gene 2-3-9 is determined for both sugarbeet genotypes 1K0088 and 4B5421. Genomic DNA is isolated from theleaves of both genotypes according to Saghai-Maroof et al., (1984). Each10 μg genomic DNA is individually cleaved by restriction enzymes EcoRI,HindIII, PstI, SalI, BamHI, EcoRV, Xhol and BglII, and the resulting DNAfragments are separated in a 0.8% agarose gel. The DNA fragments aretransferred by alkaline transfer to a hybond N nylon membrane (AmershamPharmacia Biotech, Freiburg). Radioactive labeling of 20 ng of the cDNAfragment 2-3-9 with 50 μCi ³²P-dATP (6000 Ci/mMol, Amersham PharmaciaBiotech, Freiburg) and hybridization is exactly done as described forRNA blots. After hybridization, the nylon membrane is exposed on a X-rayfilm (Kodak BioMax MS, Kodak AG, Stuttgart) in the presence of anintensifying screen (Kodak BioMax MS Intensifying Screen, Kodak AG,Stuttgart) for 16 h at −80° C., and the X-ray film is subsequentlydeveloped.

The autoradiogram of the DNA blot shows that in the genome of genotype1K0088 two copies of gene 2-3-9 are present, and that in genotype 4B5421only one gene copy is present (FIG. 4). This estimation results from theobservation that restriction digestion of the 1K0088 DNA with EcoRI andHindIII leads to three and with PstI to two hybridization signals, whileunder these conditions the DNA of 4B5421 leads to one signal with EcoRIand PstI and to two signals with HindIII.

Isolation of a Full-Length 2-1-48 cDNA Clone by RACE

Before genomic DNA fragments with the promoter region of gene 2-1-48were identified, first a potentially full-length 2-1-48 cDNA wasreconstructed. For this, the marathon cDNA amplification kit of CLONTECHwas used. Marathon cDNA amplification is a method in order to conduct a5′ and 3′ RACE (rapid amplification of cDNA ends) of one template. Intotal, six RACE products were sequenced for the 5′ region and four RACEproducts were sequenced for the 3′ region. By comparison of thesequences with each other and with the original cDNA fragment 2-1-48, asequence for a potentially full-length cDNA fragment was reconstructed.The cDNA reconstructed from RACE products has an overall length of 841base pairs and approximately corresponds to the length of the taprootspecific transcript (approx. 800 bp), which was detected by Northernblot hybridization. The translation of all three possible reading framesled to a single unspaced reading frame of 150 amino acids. All otherpossible translation products comprised numerous stop codons. The 150amino acids protein shows a homology to the 152 amino acid major latexprotein homologue from Mesembryanthemum crystallinum. 66% of amino acidsof both proteins (99 out of 149) are identical. The function of theprotein is not known. The 5′ ends of the cDNA clones of genes 2-1-36 and2-3-9 were amplified by 5′ RACE and isolated as described for gene2-1-48.

Isolation of Promoters of Genes 2-1-48 and 2-1-36

The promoter regions in genomic clones of genes 2-1-48 and 2-36 wereidentified and subcloned. For this, clones of the sugar beet genotype1K0088, which carry homologue sequences to the cDNA clones 2-1-48 and2-1-36, were isolated from a genomic bank. The bank is established inlambda vector EMBL3 SP6/T7 and comprises genomic fragments with anaverage size of 20 kb. For cloning, the genomic DNA was partiallycleaved with MboI and ligated into the BamHI restriction site of EMBL3.The inserts can be cleaved with XhoI. For the isolation of genomicclones, approx. 300,000 genomic clones were hybridized with cDNAfragments 2-1-48 and 2-1-36 in a plaque hybridization experiment(Sambrook et al., 1989). Hybridized phage plaques were picked andreplated until only the corresponding hybridized phage clone was presentin the probe. In total, thirteen clones were purified with the cDNAfragment 2-1-48, and 10 phage clones were purified with the cDNAfragment 2-1-36.

In these clones the genomic region 5′ to the reconstructed full-lengthcDNA was then amplified and subcloned. For this, a restriction cleavagesite for a restriction enzyme was searched in the cDNA, which isrelatively close to the 5′ end of the cDNA. For clone 2-1-48 a NcoIrestriction site was identified, which is approx. 175 base pairs fromthe transcription initiation. The genomic clones, which were isolatedwith the cDNA fragment 2-1-48, were cleaved with XhoI and NcoI,separated gel electrophoretically and hybridized with an radio activelylabeled oligonucleotide, which is 5′ to the NcoI restriction site. Thishybridization identified the NcoI/XhoI fragment, which comprised genomicsequences 5′ to the reconstructed cDNA. These fragments were subclonedfrom three different lambda clones (L1, L12, L9) after fill-in of theends with Klenow polymerase into a SmaI digested plasmid vector(pBluescript II SK). Bacterial colonies with recombinant plasmids wereidentified by colony hybridization. For each lambda clone three (L12-14,L12-15, L12-16, L-9-10, L-9-11, L-9-12) or two (L1-05, L1-06),recombinant plasmids were sequenced from both sites. At the ends of allclones the NcoI restriction site from the cDNA and the XhoI restrictionsite from the polylinker of the lambda vector were identified. Thesubcloned XhoI/NcoI inserts of the three lambda clones are approx. 7000(L1), 4000 (L9) and 5000 (L12), respectively, base pairs long. The 4089base pairs insert of the XhoI/NcoI fragment, which was used forsubcloning, is depicted as SEQ ID NO: 1. The nucleotides 1-3967 comprisethe entire regulatory 5′ region of the gene and therefore the promoter2-1-48. The transcribed, non-translated DNA sequence reaches fromposition 3911-3967 in comparison to the 5′ end of full-length cDNAclone. The positions 3968-4089 correspond to the 5′ end of the codingregion. The DNA sequence present in plasmid L9 differs from the DNAsequence of SEQ ID NO:1 due to the cloning technique, so that the basepair at position 1 and 4089 is missing.

From the isolated DNA of the phages, which hybridizes with cDNA clone2-1-36, a 1.923 kb NdeI-NdeI fragment, which comprises the promoter ofthe gene 2-1-36, was identified with the help of an oligonucleotidespecific for the 5′ region of the cDNA clone 2-1-36. The DNA ends of theNdeI-NdeI fragments were blunt ended by Klenow treatment, and thepromoter was cloned in the vector pBluescript II SK, digested with therestriction enzyme SmaI. The nucleotide sequence of the subclonedfragment was determined. From the analyzed sequence 1919 bp are depictedas SEQ ID NO: 2. The nucleotides 1-1840 comprise the entire subclonedregulatory 5′ region of the gene and therefore the promoter 2-1-36. Thetranscribed non-translated DNA sequence reaches from position 1606-1840,in comparison to the 5′ end of the full-length cDNA clone. The positions1841-1919 are the first 79 translated base pairs of the gene.

Isolation of Promoters of Both Genes 2-3-9

The promoter regions of gene 2-3-9, which is present in two copies inthe sugar beet genotype 1K0088, were identified and isolated. For this,lambda phages were isolated from the genomic bank of the genotype1K0088, which inserts comprise a homology to the cDNA clone 2-3-9. Intotal, 300,000 phages of the bank constructed in vector EMBL 3 SP6/T7were seeded according to the Lambda Library Protocol Handbook, Clontech,PT 1010-1 using E. coli strain K802 in melted LB top agarose+10 mM MgSO₄on 150 mm petri dishes, which comprise LB medium+10 mM MgSO₄. Theconcentration of phages per plate was 25 000. In order to screen theseeded phages for promoters of gene 2-3-9, two replica of nylon filter(NEN Life Science Prodcuts, #NEF 978Y) per plate were prepared. For thehybridization experiment, the filters were hybridized with theradioactive labeled cDNA fragment 2-3-9. Twelve positive hybridizedphages were picked with the help of a Pasteur pipette, isolated bycorresponding microbiological dilution steps and purified until purity.The purity of the phage isolates was tested by radioactive hybridizationafter each step. DNA was isolated from 9 positive lambda phages by usingthe Qiagen lambda DNA preparation kit (Qiagen, Hilden, Germany). Bycombination of long distance PCR (LD-PCR) and restriction analysis, thelocation and the orientation of the coding region of gene 2-3-9 and thusof the promoter with respect to the left and the right phage arm wasdetermined.

For the amplification of the cloned genomic fragments by LD-PCR, aprimer combination was used, in which a commercially available 5′ and 3′primer binds in the left and right phage arm, respectively, outside thecloned sugar beet DNA. The 5′ and 3′ primer of the “EMBL3 LD-InsertScreening Amplimer Set” (Clontech #9104-1, Heidelberg, Germany), whichare phage arm specific comprise the nucleotide sequence CTG CTT CTA ATAGAG TCT TGC TGC AGA CAA ACT GCG CAA C (SEQ ID NO: 5) and TGA ACA CTC GTCCGA GAA TAA CGA GTG GAT CTG GGT C (SEQ ID NO: 6), respectively. Theamplification of genomic sugar beet DNA fragments was carried out withthe help of the “Advantage Genomic PCR Kit” (Clonetech #K1906-1,Heidelberg, Germany).

The PCR conditions, using 100 ng lambda DNA, a primer concentration of0.4 μM, 0.5 μl Tth polymerase mix and 25 μl reaction volume in amulticycler PTC-200 (MJ Research, Watertown, Mass., USA) were asfollows:

1 x Step 1: 1 min 95° C. 25 x  Step 2: 15 sec 95° C. Step 3: 24 min 68°C. 1 x Step 5: 10 min 68° C.

Further, for analytical purposes the 5′ and 3′ primer of the “EMBL3LD-Insert Screening Amplimer Set” were used each in combination withprimers S82 and S83, which are specific for the 5′ and 3′ portion of thecoding region of gene 2-3-9. Thereby, the size of the genomic fragmentsadjacent to the reading frame could be determined. Primers S82 and S83comprise the sequence AGG TTA TCA AAA GGC CCC TTT CCA GTC A (SEQ ID NO:7) and GTT TGT GCA AGC CGA GCT GGT GAA CGC C (SEQ ID NO: 8). The PCRconditions correspond to the above-described LD-PCR conditions with aDNA amount reduced to 20 ng.

For restriction analysis of lambda clones with each 200-400 ng isolatedphage DNA or 200 ng LD-PCR products, were cut individually withrestriction enzymes ClaI, EcoRI, EcoRV, HindIII, PstI, SacI, SalI, XhoIand with enzyme combinations PstI/SalI and ClaI/SalI. The DNA fragmentswere separated in a 0.8% agarose gel, transferred to nylon membranes andhybridized with radioactively labeled cDNA clone 2-3-9 according toSambrook et al., 1989. The evaluation of PCR and restriction analysisshowed that 8 lambda clones contained genomic copy 1 of gene 2-3-9 andonly one clone contains copy 2 of gene 2-3-9.

Subcloning and Characterization of the C1 Promoter, which is Active inAbove-Ground Organs

Starting from 8 lambda clones, which carry genomic copy 1 of gene 2-3-9,the phage γ c6.1.1 was chosen for subcloning of the promoter. In thefollowing, promoter of copy 1 of gene 2-3-9 is termed C1 promoter. Thefull-length coding region of the gene and the C1 promoter and theregulatory 3′ region of the gene are located on a 6.294 kb ClaI-ClaIfragment. The ClaI-ClaI fragment was isolated from the DNA of phage λc6.1.1 and subcloned into vector pBluescript II KS+ (Stratagene,Heidelberg, Germany), which was ClaI cleaved and dephosphorylated withalkaline phosphatase treatment. The resulting plasmid is termed pc1a.The nucleotide sequence of the subcloned fragment with the genomicfragment of copy 1 of gene 2-3-9 was determined. 1148 bp of thedetermined nucleotide sequence are depicted as SEQ ID NO: 3. Nucleotides1-1097 comprise the entire regulatory 5′ region of the gene located onplasmid pc1a, and therefore comprise the C1 promoter. Comparison withthe 5′ end of the respective cDNA revealed that the transcribed,non-translated DNA sequence extends from position 984-1097. The position1098-1148 are the first 51 translatable base pairs of the gene. Thegenomic 5′ region following the C1 promoter was also subcloned fromphage γ c6.1.1. For this, the phage DNA was cleaved with restrictionenzymes SalI and ClaI, the isolated 6.026 kb SalI-ClaI fragment wassubcloned into the corresponding restriction sites of vector pBluescriptII KS+. The resulting plasmid is termed pc1b (FIG. 5).

Fusion of Sugar Beet C1 Promoter with the Luciferase Gene from Photinuspyralis

In order to determine the activity of the isolated C1 promoter in sugarbeet leaves, the C1 promoter was translationally fused to the luciferasegene from Photinus pyralis. The reporter gene vector pGEM-luc (Promega,Mannheim, Germany), which carries the P. pyralis luciferase gene, wasconnected with the regulatory 3′ region of the nopaline synthase (nos)gene, in order to obtain a vector suitable for expression in plants. Forthis, vector pBI101.3 (Clontech, Heidelberg, Germany) was linearizedwith EcoRI, and EcoRI DNA ends were modified by Klenow treatment toblunt DNA ends. By re-cleaving with SacI, the 0.26 kb nos terminator wasset free, and then isolated. Vector pGEM-luc was linearized withrestriction endonuclease SfiI, and the restriction site was blunted byT4 polymerase treatment. After restriction of the thus pre-treatedvector with restriction enzyme SacI, the isolated 0.26 kb nos terminatorwas inserted as EcoRI (filled-in)-SacI fragment in pGEM-luc. Theresulting vector is termed pluc-nos2 (FIG. 6). The C1 promoter fragmentfrom position 1-1145 (SEQ ID NO: 3) was cleaved from the plasmid pc1ausing the restriction enzymes SalI and AviII, DNA ends were blunted byKlenow treatment and the promoter fragment was isolated. Vectorpluc-nos2 was linearized with restriction enzyme ApaI, and the DNA endswere blunted by T4 polymerase treatment. After dephosphorylation of thevector, the C1 promoter was subcloned as SalI (filled-in)-AviIIfragment. The resulting vector is termed pc1L-1097 (FIG. 7). In vectorpc1L-1097, the promoter is connected translationally with the luciferasegene by the base pairs of gene 2-3-9, coding for the first 16 aminoacids. In order to answer the question, whether the C1 fragment used inpc1L-1097 is sufficient for promoter activity, this regulatory DNAfragment was enlarged by the adjacent genomic 5′ region from sugar beet.For this, the 6029 bp DNA fragment from pc1b was used. Plasmid pc1b wascleaved with restriction enzyme KpnI, and the DNA ends were blunted byT4 polymerase treatment. By recleavage with the restriction enzyme NotI,the genomic region could be isolated as KpnI (blunted)-NotI fragment,and could be cloned into vector pc1L-1097. The vector pc1L-1097 hadfirst been linearized with HindIII, the DNA ends were blunted withKlenow fragment, and were then again treated with restriction enzymeNotI. The resulting plasmid is termed pc1L-7126 (FIG. 8) and comprisesthe 5′ region of copy 1 of gene 2-3-9 from position 1-7126.

Detection of C1 Promoter Activity in Sugar Beet Leaves by BallisticTransformation

The activity of the reporter gene constructs pc1L-1097 and pc1L-7126 wasmeasured in leaves after ballistic transformation. Ballistictransformation was carried out with a PDS-1000/He Particle DeliverySystems (BioRad) in accordance with the manufacturer's recommendations.As a microcarrier, gold powder type 200-03 (Heraeus, Hanau, Germany)with a particle size of 1.09-2.04 μm was used. Preparation and loadingof the microcarrier with the reporter gene constructs was carried outaccording to BioRad protocol US/EG Bulletin 1688. The vectors pc1L-1097and pc1L-7126 were used in equimolar amounts. In order to avoid resultvariations, which are based on different transformation efficiencies andnot on differences of the strength of promoters, a normalization of thegene expression was conducted. For this, plasmid p70Sruc with theluciferase gene from Renilla reniformis as a second reporter gene systemin a volume amount of 7:3 was mixed with vectors pc1L-1097 andpc1L-7126, respectively, and used for loading of the microcarrier. Themeasurement of a further reporter gene activity, which expression isunder the control of the double 35S promoter, allows using this resultas reference for the determination of the transformation efficiency ofthe single experiment.

For each of the reporter gene constructs to be tested, three shootingexperiments were conducted, the gene expression was normalized, andthen, a mean value of the normalized gene expression was calculated. Forcontrol purposes two shooting experiments were carried out with goldpowder without DNA loading. Thus determined enzyme activities representendogenous background activity in leaves, and were subtracted fromfurther experimental values. Per shooting experiment, 13 leaf rondelswere punched out with a cork drill (size 8) from young and old sugarbeet leaves, and osmotically pretreated for 6 h in 90 mm petri dishes onMS medium+0.4 M mannitol solidified with agar at 25° C. Shootingparameters were 1550 psi burst disk size, 9 cm distance between leafrondels and burst disk, and 27.5 in Hg negative pressure in the toolchamber. After shooting, the plates were incubated for 16 h at 25° C.under light.

Photinus and Renilla luciferace activity was determined with the dualluciferace reporter assay system (Promega, Mannheim, Germany) in a Lumat9501 luminometer (PE Biosystem) according to the manufacturer'sinstructions. An enzyme extract suitable for measurement was obtained bymaceration of the leaf rondels of one shooting experiment in liquidnitrogen. After vaporization of nitrogen, the powdery leaf material washomogenized with 10× volume (v/w) of passive lysis buffer (PLB). Theliquid supernatant was transferred to a 1.5 ml Eppendorf tube andcentrifuged for 5 min at 4° C. and 20,000 g. The clear supernatant wascollected, and 10 μl raw extract was used for Photinus and Renillaluciferase activity measurement, respectively.

The mean value of the normalized gene expression for construct pc1L-1097 was 8.0 for small leaves and 9.6 for large leaves (table 4). Forconstruct pc1 L-7126, the mean value of the normalized gene expressionwas 3.2 for small leaves and 7.0 for large leaves (table 4). Therefore,the shorter C1 promoter fragment of plasmid pc1L-1097 in beet leaves isnot only sufficient for the expression of the reporter gene, but it isalso more active than the longer C1 promoter fragment of constructpc1L-7126.

Subcloning and Characterization of the C2 Promoter, which is Active inAbove-Ground Organs

Subcloning of the promoter of the second copy of gene 2-3-9 resultedfrom phage λ c7.2.1. In the following, the promoter of this gene isindicated as C2 promoter. The preceding restriction analysis had shownthat the 5′ region of the coding region and approx. 3.0 kb of theregulatory 5′ region of the second copy of gene 3-2-9 are located on a4,002 kb PstI-PstI fragment of phage γ c7.2.1.

The 4,002 kb PstI-PstI fragment of phage λ c7.2.1 was isolated afterPstI restriction digestion and cloned into vector pBluescript II KS+(Strategene, Heidelberg, Germany). For this, vector pBluescript II KS+was also cleaved with restriction enzyme PstI and then dephosphorylatedby treatment with alkaline shrimps phosphatase (Roche Diagnostics GmbH,Mannheim, Germany). After ligation and transformation into E. colistrain XL-1, plasmid DNA was isolated from E. coil transformants, andpositive clones were identified by restriction analysis. The resultingplasmid is termed pc2. The nucleotide sequence of the 4,002 kb PstI-PstIfragment was determined. 3049 bp of the nucleotide sequence are depictedas SEQ ID NO: 4. Nucleotide 1-2998 comprise the entire regulatory 5′region of the gene located on plasmid pc2, and therewith the C2promoter. The transcribed, non-translated DNA sequence, compared to the5′ end of the respective cDNA, extends from position 2928-2998. Position2999-3049 are the first 51 translated base pairs of the gene.

Fusion of Sugar Beet C2 Promoter with the Luciferase Gene from Photinuspyralis and Preparation of Deletion Constructs

In order to detect the activity of the isolated C2 promoter in sugarbeet leaves, the C2 promoter was translationally fused to the luciferasegene from Photinus pyralis. For this, the 4002 bp DNA fragment subclonedin pc2, comprising the C2 promoter and the 5′ region of the codingregion of the second copy of gene 2-3-9 was isolated after restrictiondigestion with enzymes NotI and EcoRI. The DNA fragment was thenpartially cleaved with enzyme AviII, and an approx. 3100 bp NotI-AviIIfragment was isolated, which comprises base pairs 1-3046 of SEQ ID NO:4. The NotI-AviII fragment was then subcloned into reporter gene vectorpluc-nos2. For this, vector pluc-nos2 was first cleaved with restrictionenzyme ApaI, and the sticky ends were blunted by T4 polymerasetreatment. By recutting with restriction enzyme NotI, vector pluc-nos2was transferred into a condition susceptible for receiving theNotI-AviII fragment. The resulting fragment is termed pc2L-2998 (FIG.9). Vector pc2L-2998 carries the C2 promoters sequence of SEQ ID NO: 4from nucleotide position 1-2998 and the first 48 translated base pairsof gene 2-3-9 from position 2999-3046.

In order to identify the minimal DNA fragment size, which is necessaryfor the activity of the C2 promoter, three 5′ deletion constructs wereprepared. Constructs pc2L-1827, pc2L-989 and pc2L-342 comprise the C2promoter sequence of SEQ ID NO: 4 from nucleotide position 1172-2998,2011-2998 and 2657-2998, respectively, and the first 48 translated basepairs of gene 2-3-9 from position 2999-3046 (FIG. 10-12). Based onpc2L-2998, the vectors were developed in detail as follows:

Vector pc2L-1827 by a restriction digestion with enzymes KpnI and NotI,a subsequent blunting of the DNA ends with T4 polymerase treatment andreligation of the vector.

Plasmid pc2L-989 by a digestion with restriction enzyme SmaI andreligation of the vector.

Plasmid pc2L-342 by a digestion with restriction enzyme NotI and apartial SalI digest. After blunting of the DNA ends by Klenow treatment,the vector was religated.

Detection of C2 Promoter Activity in Sugar Beet Leaves by BallisticTransformation

The activity of reporter gene constructs pc2L-2998, pc2L-1827, pc2L-989and pc2L-342 was determined in leaves after ballistic transformation.Ballistic transformation and determination of reporter gene activitieswere done as previously described for the C1 promoter. Vectorspc2L-2998, pc2L-1827, pc2L-989 and pc2L-342 were used in an equimolarratio. In order to avoid result variations, which are based on differenttransformation efficiencies and not on differences in promoteractivities, a normalization of the gene expression was done with thehelp of plasmid p70Sruc. Plasmid P70Sruc carries the luciferase genefrom Renilla reniformis as a second reporter gene system. For eachreporter gene construct to be investigated, four shooting experimentsper DNA preparation were conducted. For constructs pc2L-2998 andpc2L-1827 one DNA preparation was used and for constructs pc2L-989 andpc2L-342 two DNA preparations were used. The measured gene expressionwas normalized and then, a mean value of the normalized gene expressionwas calculated. For control purposes four shooting experiments with goldpowder without DNA loading were conducted. The mean value of thenormalized gene expression for constructs pc2L-2998, pc2L-1827, pc2L-989and pc2L-342 was in detail 8.0, 4.5, 6.45 and 6.45, respectively (FIG.13). Therefore, all C2 promoters fragments are suitable to allow theexpression of a gene in leaves. The smallest promoter fragment ofconstruct pc2L-342, which refers to nucleotide sequence of SEQ ID NO: 4from position 2657-3046, is equally active as the larger promoterfragments of constructs pc2L-2998, pc2L-1827 and pc2L-989.

Construction of Plant Transformation Vectors pc1G-1097 and pc2G-2998 asan Example

For the stable transformation of gene fusions of C1 and C2 promoters,active in above-ground organs, and the gus reporter gene, binary vectorspc1 G-1097 (FIG. 14) and pc2G-2998 (FIG. 15), were developed.

For construction of pc1G-1097, the C1 promoter is isolated from vectorpc1 L-1097 as approx. 1.17 kb HindIII-BamHI fragment and inserted intothe HindIII and BamHI linearized binary vector pBI101.3 (Clontech,Heidelberg). Subcloned C1 promoter comprises the nucleotide sequence ofSEQ ID NO: 3 from position 1-1145. In the resulting vector pc1G-1097,the C1 promoter is linked translationally with the gus gene frompBI101.3 by the base pairs coding for the first 16 amino acids of gene2-3-9.

Vector pc2G-2998 is constructed in such a way that the C2 promoter isisolated from vector pc2L-2998 and translationally linked to the gusgene from pBI101.3. Vector pc2G-2998 carries, like plasmid pc2L-2998,the C2 promoter sequence of SEQ ID NO: 4 from nucleotide position 1-2998and the first 48 translated base pairs of gene 2-3-9 from position2999-3046. For this, pc2L-2998 was cleaved with restriction enzyme PstI,and the DNA ends were blunted by Klenow treatment. After treatment withrestriction enzyme BamHI, the C2 promoter could be isolated as approx.3070 bp DNA fragment and cloned into adequately prepared binary vectorpBI101.3. For insertion of the C2 promoter, vector pBI101.3 wasinitially linearized with restriction enzyme SalI, and the DNA ends werefilled-in by Klenow treatment. The vector was then cleaved with enzymeBamHI.

Transformation of Constructs pc1G-1097 and pc2G-2998 in Plants

The constructs defined for the production of transgenetic plants areinitially transferred into Agrobacterium tumefaciens strain GV2260 by adirect DNA transformation method (An, 1987). The selection ofrecombinant A. tumefaciens clones is carried out by using the antibiotickanamycin (50 mg/l). In the following, as an example, the transformationfor vector pc1G-1097 is described.

The reporter gene cassette consisting of the translational fusionbetween the C1 promoter and the gus gene is transformed into summerbrassica genotype Drakkar with the help of A. tumefaciens according toHorsch et al., (1985). Transgenetic plants are selected with theantibiotic kanamycin. The presence of the promoter in transgeneticplants can be verified by PCR. The use of primers GTGGAGAGGCTATTCGGTA(SEQ ID NO: 9) and CCACCATGATATTCGGCAAG (SEQ ID NO: 10) leads to theamplification of a 535 bp DNA fragment from the nptII gene. PCR is donewith 10 ng genomic DNA, a primer concentration of 0.2 μM at an annealingtemperature of 55° C. in a multicycler PTC-200 (MJ Research, Watertown,USA).

Using the above described techniques, twenty independent brassica andtobacco transformants were obtained with binary vector pc1G-1097,respectively, and twenty independent brassica and potato transformantswere obtained with binary vector pc2G-2998, respectively.

Determination of C1 and C2 Promoter Activity in Leaves of TransgeneticBrassica, Potato and Tobacco Plants

In order to determine the activity of C1 and C2 promoters in leaves oftransgenetic brassica and tobacco plants, a histochemic GUS staining wasdone. Leaves are taken from transgenetic and non-transgenetic in vitroplants, vacuum infiltrated with GUS staining solution (2 mM5-bromo-4-chloro-3-indoyl-beta-glucuronide, 50 mM sodium phosphate pH7.0, 0.5% Triton X-100, 2% N,N-dimethylformamide) for 15 sec, and thenincubated for 16 h at 37° C. Then, chlorophyll from leaves is extractedwith 70% ethanol. Blue staining of the tissue indicates regions in whichGUS activity is present and the promoter is expressed.

Leaf portions from brassica transformant pc1G-1097-86 and tobaccotransformant pc1G-1097-3 show an intensive even blue staining of thetissue (FIGS. 16 and 17). In comparison thereto, leaf portions ofnon-transgenetic brassica and tobacco plants are white after thistreatment. Also intensively blue stained are leaf portions of potatotransformant pc2G-2993-1. These results show that C1 and C2 promoters instable transformed brassica, potato and tobacco plants are functional,and that the gus reporter gene under the control of the promoter isexpressed in the family of Brassicaceae and Solanaceae.

Homology Between C1 and C2 Promoters

Nucleotide sequence comparison between DNA sequences of promoters C1 andC2 shows that the promoter region of the C1 promoter from position780-1051 with the exception of three base pairs is entirely identicalwith the sequence of the C2 promoter from position 2707-2984 (FIG. 19).The sequences of the C1 promoter from position 1-799 do not show anysignificant homology to the DNA sequence of the C2 promoter fromposition 1-2706. The homologue region between the promoters comprisespositions from −320 to −42 of the C1 promoter and the positions from−292 to −21 of the C2 promoter with respect to the translation start. Inthe homologue promoter region next to the TATA box and the respectivetranscription start are multiple cis-elements, which are conservedbetween both promoters. In C1 promoter, the TATA box extends fromposition 950-956 and in C2 promoter from position 2877-2883. Next to theTATA box, among the elements, which are frequently found in promoters,are a CAAT box at position 884-887 in C1 promoter and at position2811-2814 in C2 promoter. Among the cis-elements specific for C1 and C2promoters are a 4× repeat of the GATA box at positions 812-815, 820-823,832-835 and 838-841 in the C1 promoter and at the positions 2739-2742,2747-2750, 2759-2762 and 2765-2768 in the C2 promoter. The GATA box wasidentified in the 35S promoter as binding site for the ASF-2transcription factor and is present in a 3× repeat in the lightregulated promoter of the chlorophyll a/b binding protein of petunia(Lam and Chua, 1989). At position 832-837 of the C1 promoter and atposition 2759-2764 of the C2 promoter is an I box with the sequenceGATAAG. The I box was first described for the light regulated rbcSpromoters of tomato and arabidopsis (Giuliano et al., 1988). Atpositions 844-849 and 985-990 of the C1 promoter and positions 2771-2776and 2912-2917 of the C2 promoter a 2× repeat of the GT-1 binding site islocated. The GT-1 binding site with the consensus sequenceG(A/G)(A/T)AA(A/T) was described for promoters of several light inducedgenes and the pathogen resistance gene PR-1 (Zhou, 1999). A 2× repeat ofa DNA sequence, which is important for circadian expression is locatedat positions 913-922 and 1014-1023 in the C1 promoter and at thepositions 2840-2849 and 2941-2950 in the C2 promoter. The circadian boxwith the consensus sequence CAANNNNATC (SEQ ID NO: 11) was identified ina light regulated Lhc promoter of tomato (Piechulla et al., 1998).

Change of the Carbohydrate Metabolism of Plants by Use of Root SpecificPromoters 2-1-48 and 2-1-36, Exemplified by Avoidance of StorageSubstance Loss

The carbohydrate metabolism of plants can specifically be improved bythe use of root specific promoters of genes 2-1-48 and 2-1-36. As anexample, the expression of the invertase inhibitor gene from tobacco(Greiner et al., 1998) in the root of sugar beets under control of rootspecific active promoters 2-1-48 and 2-1-36, respectively, is described.By root specific expression of the storage substance losses, which arepresent after the root harvest until root processing, sucrose and sugar,technically undesired accumulation of glucose and fructose, is reduced,and thereby the sugar yield is altogether improved. Using a rootspecific promoter allows, in contrast to a promoter, which isconstitutively active in all tissues, to restrict the expression of theinvertase inhibitor gene to the root. By this regional restriction,undesired yield-reducing effects, which would be present if theinhibitory gene were expressed in all plant parts, are avoided.

Promoters 2-1-48 and 2-1-36 can be connected to the tobacco invertaseinhibitor gene, respectively, as translational or transcriptional fusionand transferred by A. tumefaciens mediated transformation into sugarbeet. For this purpose the respective binary vectors are transformedinto A. tumefaciens isolate C58 ATHV according to An (1987). The plantstarting material for transformations of sugar beets are seedlings. Forthis, seeds of the sugar beet are surface disinfected with 12% sodiumhypochloride, and then germinated for 3-4 weeks under sterileconditions. The seed leaves of these seedlings are then cut with thehelp of a scalpel and incubated for 5-10 min in a diluted overnightculture of A. tumefaciens isolates (OD 0.2-0.3). Then plant parts areswap-dried and cocultured for 3 days on solid 1/10 MS medium+30 g/lsucrose. After the cocultivation phase, the explants are transferred toselection medium (MS medium+0.25 mg/l BAP, 0.05 mg/l NAA), 500 mg/lbetabactyl, 20 g/l sucrose). For selection of transformants kanamycin isused. After 7-12 weeks, transgenetic plants develop, which can bepropagated and rooted.

In order to detect the transgenetic character of the plants molecularbiologically, the nptII gene transferred to the plants is detected asdescribed by two gene specific primers. Expression of the invertaseinhibitor gene in sugar beet roots is detected by RNA blot studies. Forthis, clone plants starting from primary transformants andnon-transgenetic control plants are produced and transferred into agreenhouse for further culturing. RNA is isolated from leaves, petiolesand the root of invertase inhibitor and control plants according toLogemann et al. 1987, as previously described, gel-electrophoreticallyseparated and transferred to a nylon membrane. The subsequenthybridization with the invertase inhibitor gene from tobacco shows thatthe gene is only expressed in the root and not in above-ground organs ofthe transgenetic plants. In order to analyze the positive effect to theroot specific expression, a storage experiment is conducted.

The fully developed storage roots from 24 week old inhibitor and controlplants are harvested and superficially injured by a 30 seconds treatmentin a commercially available cement mixer (Attika) in order to createinjuries typical for mechanical beet harvest. Subsequently the beets arestored at 17° C. and 27° C. From plant material stored at 17° C. 1, 3, 47, 14, 21, 28, 35 and 46 days after harvest and from the beets stored at27° C. 1, 3, 4, 7 and 14 days after harvest 5 beets are withdrawn,respectively. The beets are homogenized and the content of fructose,glucose and sucrose is determined. Non-transgenetic sugar beets storedat 17° C. and 27° C. show, beginning at the fourth storage day, asignificant increase of glucose and fructose content and a decrease ofsucrose concentration. In contrast, the storage roots of the invertaseinhibitor plants comprise less accumulation of glucose and fructose andless decrease of sucrose concentration in comparison to the controlplants.

Change of the Carbohydrate Metabolism of Transgenetic Roots by Use ofthe Root Specific Promoters 2-1-48 and 2-1-36 Exemplified by theReduction of Wound Induced Vacuolar Invertase Activity

The described improvement of the carbohydrate metabolism of plants canalso be detected by transgenetic root cultures (“hairy root”) of thesugar beet. Three A. tumefaciens C58 ATHV derivatives, which aretransformed with the 2-1-48 promoter inhibitor construct, the 2-1-36promoter inhibitor construct or only with the parent vector,respectively, are grown for 24 h in liquid LB medium+50 mg/l kanamycin.In parallel, Agrobacterium rhizogenes strain 15834 is cultured in liquidTSB medium+25 mg/l rifampicin. Subsequently, A. tumefaciens and A.rhizogenes strain are cultured for 21 h in the respective medium withoutantibiotics. Optical density of the bacterial cultures is determined andadjusted to A₆₀₀=0.4-0.6. Leaf stalks of 3-4 week old sugar beets, whichwere cultured under in vitro conditions, are cut into 0.5 cm portionsand shortly dipped in a 1:1 mixture of the A. tumefaciens and A.rhizogenes cultures. The leaf portions are cocultered with the bacteriafor 2 days under constant light and 25° C. on solid MS medium+0.5 mg/lBAP. After cocultivation, the stem segments are transferred to solid MSmedium+0.5 mg/l BAP+350 mg/l betabactyl (SmithKlineBeecham)+150-300 mg/lkanamycin, and incubated under weak light. After approx. 12 days, thefirst transgenetic roots are visible, which are cut-off and propagatedon ½ B5 medium+300 mg/l betabactyl+300 mg/l kanamycin. The transgeneticroot cultures are propagated for further experiments on ½ B5 mediumwithout antibiotics.

The expression of the invertase inhibitor gene from tobacco intransgenetic root cultures is detected by an RNA blot experiment. RNA isisolated from root cultures, which were transformed with the 2-1-48promoter inhibitor construct, the 2-1-36 promoter inhibitor constructand the parent vector, respectively. The RNA is gel-electrophoreticallyseparated, blotted, and the nylon filter is hybridized with aradioactively labeled invertase inhibitor gene as probe. Thehybridization result shows that the invertase inhibitor gene of tobaccois only expressed in root cultures transformed by the 2-1-48 promoterinhibitor construct and the 2-1-36 promoter inhibitor construct, but notin root cultures transformed by the parent vector for control purposes.

The improvement of the carbohydrate metabolism of root cultures isproven by the determination of wound induced vacuolar invertaseactivity. The acid vacuolar invertase of the sugar beet is localized inthe vacuole, like sucrose. During the first 12 days after beet harvestan increase of the inverted sugar concentration and of activity of theacid vacuolar invertase is observed. The wounding of the beet body bydecapitation of the sugar beet and the subsequent harvest procedure andthe interruption of the vegetation period are regarded as the reasonsfor the induction of the acid invertase activities and therewith theincrease of inverted sugar in the beet (Berghall et al., 1997).

By root specific expression of tobacco invertase inhibitor gene intransgenetic root cultures, the activity of vacuolar invertase inreaction to the wound stimulus is strongly induced and the formation ofinverted sugar is dramatically reduced. Root cultures, which weretransformed with the 2-1-48 promoter inhibitor construct, the 2-1-36promoter inhibitor construct and the parent vector, respectively, arecut with a scalpel into 3 mm portions and are incubated for 24 and 48 hin liquid ½ B5 medium. Subsequently, the roots are homogenized and theactivity of acid vacuolar invertase is determined. Cultures, in whichthe inhibitor is root specifically expressed by promoters 2-1-48 and2-1-36 show in reaction of the wounding significantly less activity ofacid vacuolar invertase and therewith an improved carbohydratemetabolism in comparison to control probes.

Change of the Carbohydrate Metabolism of Plants by Using the RootSpecific Promoters 2-1-48 and 2-1-36 Exemplified by the Synthesis of NewCarbohydrates

The carbohydrate metabolism of plants can be significantly improved bythe use of root specific promoters of genes 2-1-48 and 2-1-36 in orderto produce new carbohydrates. As an example, the synthesis of the sugarsubstitute palatinose and the sweetener palatinite in roots of sugarbeets under the control of root specifically active promoters 2-1-48 and2-1-36 is described.

Palatinite (glucosyl-α-(1,6)-sorbit/mannit) can be synthesized startingfrom sucrose in two reaction steps. Sacchrose-6-glucosylmutase catalysesthe conversion of sucrose into palatinose (isomaltulose). Palatinose isreduced by sorbit dehydrogenase into palatinite (isomalt).

The combination of root specific promoters 2-1-48 and 2-1-36 with afusion between the vacuolar transit peptide of potato palatine gene andthe gene for saccharose-6-glucosylmutase from Pseudomonas mesoacidophilaor Protaminobacter rubrum (Klein, 1995), respectively, allows theproduction of transgenetic plants, which produce palatinose specificallyin roots only. These constructs are transformed, as previouslydescribed, in plants like sugar beet, which incorporate the storagesubstance sucrose in root vacuoles. As a selection marker the nptII genein combination with the antibiotic kanamycin is used. The transgeneticcharacter of sugar beets identified by kanamycin selection, is verifiedby PCR, using primers specific for the nptII gene.

The concentration of palatinose in roots of different transformants isdetermined by HPLC. The probes can be separated by application of therunning agent 0.1 M NaOH on a Hamilton RCX-10 (250×4.1 mm) column. Thequantification of palatinose in root extracts is done by referring topalatinose references of known concentration. Using this analyticaltechnique, sugar beet transformants can be identified, which producepalatinose in roots.

For the preparation of palatinite producing plants, palatinose sugarbeets are selected, which accumulate the highest concentration ofpalatinose in the root. These transformants are transformed once againwith a construction, which comprises the 2-1-48 and 2-1-46 promoter incombination with the sorbit dehydrogenase gene, respectively. Therefore,the dehydrogenase gene is on the one hand fused to a vacuolar transitsequence or free of a signal sequence, so that the gene product islocalized in the cytoplasm. For the selection of double-transformants abinary vector is used, which either comprises the pat or the CP4 gene sothat basta or roundup can be used as selection agent. Transformants,which comprise such a herbicide resistance are additionally molecularbiologically characterized by PCR by determining the presence of the barand the CP4 gene, respectively.

The synthesis of palatinite in transgenetic plants is quantitativelydetermined by HPLC. These investigations show that the coexpression of abacterial sacchrose-6-glucosylmutase gene in combination with a sorbitdehydrogenase gene, using root specific promoters, leads to theformation of palatinite. Only the use of root specific promoters allows,in comparison to constitutive promoters, to obtain transformants, whichshow a normal phenotype and produce palatinite without undesiredphysiological amendments in commercially interesting concentrations.

Variation of the Carbohydrate Metabolism of Transgenetic Roots by UsingRoot Specific Promoters 2-1-48 and 2-1-36, Exemplified with theSynthesis of New Carbohydrates

An improvement of the carbohydrate metabolism of plants by synthesis ofnew carbohydrates can also be demonstrated by transgenetic root cultures(“hairy root”) of sugar beet. For this purpose, the previously describedexpression cassettes consisting of the 2-1-48 and 2-1-36 promoter andthe fusion between the transit sequence of the palatine gene with thegene for saccharose-6-glucosylmutase from Pseudomonas mesoacidophila andProtaminbacter rubrum and the 2-1-48 and 2-1-36 promoter and the sorbitdehydrogenase gene, respectively, are integrated into the binary vectorBIN19 (Clontech, Heidelberg, Germany). The resulting constructs aretransformed into A. tumefaciens strain C58 ATHV. By cotransformationwith A. rhizogenes strain 15834, the gene combination ofsaccharose-6-glucosylmutase and sorbit dehydrogenase is transformed, asdescribed, in transgenetic sugar beet roots,

The quantitative detection of palatinite is done by HPLC, as described.These analytical analyses show that the expression of thesaccharose-6-glucosylmutase and sorbit dehydrogenase gene under thecontrol of root specific promoters 2-1-48 and 2-1-36 leads to theformation of the new carbohydrate palatinite in transgenetic rootcultures.

Variation of the Carbohydrate Metabolism of Plants by Using RootSpecific Promoters 2-1-48 and 2-1-36, Exemplified by the Synthesis ofNovel Polymers

The carbohydrate metabolism of plants can be specifically improved byusing root specific promoters of genes 2-1-48 and 2-1-36 by theproduction of new polymers.

The formation of new polymers can, for example, occur in roots of sugarbeets by expression of a fructan-fructan-fructosyl transferase, asucrose-sucrose-fructosyl transferase, a levan sucrase, asucrose-fructan-6-fructosyl transferase and a fructosyl transferase.

The coding regions of the enzymes are each connected with the rootspecific promoter of genes 2-1-48 and 2-1-36 and transformed into sugarbeets with the help of A. tumefaciens according to techniques known inthe art, and transferred by cotransformation with A. rhizogenes intransgenetic root cultures, respectively. The expression of transferredgenes is determined on the one hand by RNA blot studies using codingregions as hybridization probes and on the other hand by enzymaticactivity measurement. By sugar analytical measurements, transformantscan be identified, which comprise the desired polymers in highestconcentration.

Variation of the Nitrogen Metabolism of Plants by Using Root SpecificPromoters 2-1-48 and 2-1 -36 and C1 and C2 Promoters, Respectively,Active in Above-Ground Organs

The nitrogen metabolism of plants can be improved in various aspects byusing root specific promoters of genes 2-1-48 and 2-1-36 and by using C1and C2 promoters, which are active in above-ground organs. The root andleaf specific increase of the number of suitable transport proteinsimproves the uptake and the transport of N-compounds in the plant.

By using root specific expression of transporter protein genes forammonium (NH₄ ⁺) nitrate (NO₃ ⁻) and nitrite (NO₂ ⁻) ions, the nitrogenuptake from the ground can be increased and the use of N-fertilizer canbe improved. The leaf specific expression of nitrate and nitritetransport proteins serves for an efficient use of N-compounds alreadytaken up into the root by using promoters C1 and C2, which are active inabove-ground organs. The leaf specific expression of nitrate transportproteins leads to an increased phloem discharge of nitrate ions and toan increased nitrate uptake into leaf parenchyma cells. The N-reductionin plastids is increased by elevated nitrate accumulation in leafparenchyma cells. The elevated transport of nitrite from the cytosolinto the plastids by leaf specific expressions of suitable nitritetransport protein also leads to an increase amino acid biosynthesis.

Increase of Tolerance Towards Pathogens by Using Root Specific Promoters2-1-48 and 2-1-36 and/or of Promoters C1 and C2, which are Active inAbove-Ground Organs

Root specific promoters 2-1-48 and 2-1-36 and promoters C1 and C2, whichare active in above-ground organs can be used for the development offeatures, which improve the resistance or tolerance towards pathogens.

Increase of Tolerance Towards Phytopathogenic Viruses by Use of RootSpecific Promoters 2-1-48 and 2-1-36 and/or Promoters C1 and C2, whichare Active in Above-Ground Organs

Numerous phytopathogenic viruses of the sugar beet show organspecificity, i.e. the viral multiplication does usually not occur in theentire plant, but only in one specific organ or tissue type. Also,damages, which are induced by the viral infection are generallyrestricted to the infected organ. Viral pathogens of the sugar beet withorgan specificity are, e.g. BNYVV with a preference for root and BMYVand BYV with a restriction to beet leaves.

Root specific promoters 2-1-48 and 2-1-36 can be used in order todevelop a root specific BNYVV resistance in sugar beet. For this, forthe realization of gene silencing dependent virus resistance strategy, anative or mutagnized DNA partial sequence of the viral BNYVV genome iscombined with the 2-1-48 or 2-1-36 promoter. The combination between thepromoter sequence and the viral DNA sequence is designed such that thetranscription of the BNYVV sequence leads to a gene silencing, effectiveagainst BNYVV. The efficiency of this approach is determined by thedetermination of the virus titer in plants by using an ELISA test, whichis directed against the core protein of BNYVV.

Increase of Tolerance Towards Phytopathogenic Nematodes by Using RootSpecific Promoters 2-1-48 and 2-1-36

The root specific activity of promoters 2-1-48 and 2-1-36 can be used toinduce in plants as sugar beet a resistance towards nematodes likeHeterodera schachtii or in potatoes a resistance towards Globoderapallida or Globodera rostochiensis.

For this purpose, a nematode resistance gene or a gene for a nematocidacting compound is translationally or transcriptionally fused withpromoter 2-1-48 and 2-1-36, respectively, and inserted in a binary planttransformation vector like, e.g. BIN19. Nematode resistance genes, whichmediate a resistance towards Heterodera schachtii, are Hs1^(pro-1) fromBeta procumbens (Cai et al., 1997) and in the case of Globodera pallidathe Gpa2 gene of potato (Van der Vossen et al., 2000).

By A. tumefaciens mediated transformation, the promoter genecombinations are used in an already described manner for the yield oftransgenetic sugar beets or potatoes. Further, the gene constructs canbe transferred into transgenetic root cultures of the sugar beet bycotransformation using A. tumefaciens and A. rhizogenes according to thedescribed protocol. The transgenicity of produced plants is molecularbiologically detected by PCR by amplification of the nptII gene. Theroot specific expression of the resistance mediating factor, as, e.g.Hs1^(pro-1) gene, is proven by an RNA blot study using the Hs1^(pro-1)gene as hybridization probe. The resistance of the transgenetic plantsand transgenetic root cultures is examined and determined by a nematoderesistance test. The realization of nematode resistance testing with H.schachtii on transgenetic root cultures of sugar beet is described byCai et al., (1997). The person skilled in the art will find theexperimental description of the resistance test of in vitro potatoestowards G. pallida or a reference to the realization of greenhousetestings in (Van der Vossen et al., 2000).

The advantages of root specific expression of the nematode resistancegene and the nematocid component are the high resistance and the factthat the resistance mediating gene products are only produced in theorgan to be protected. The absence of the resistance mediating geneproduct in plant parts, which are intended for consumption, like thepotato bulb, increases the social acceptance and thereby the chances forsales of the transgenetic plant and the product derived thereof.

Increase of Tolerance of Transgenetic Plants Towards PytophatogenicFungi by Use of Root Specific Promoters 2-1-48 and 2-1-36 and/orPromoters C1 and C2, Which are Active in Above-Ground Organs

Root specific promoters 2-1-48 and 2-1-36 and promoters C1 and C2, whichare active in above-ground organs, can be used to develop a direct orindirect antifungal effect in plants in combination with a gene or genecombination. The antifungal effect leads to the feature of increasedfungus resistance or fungus tolerance.

The root specific promoters 2-1-48 and 2-1-36 and the promoters C1 andC2, which are active in above-ground organs, are translationally ortranscriptionally fused with genes of pathogen defense, respectively,which gene products have a direct antifungal activity. The promoter genecombinations are cloned into the binary transformation vector BIN19 andtransformed into sugar beet, potato and brassica by A. tumefaciensmediated transformation. The transgenicity of the plants is tested byPCR, as described, and the expression of the gene in roots or leaves isverified by RNA blot studies. Increased fungus resistance of plants isobserved in fungus resistance assays, as they are exemplarily describedin the following for resistance testing of sugar beets towardsCercospora beticola.

For infection of sugar beets with the leaf spot pathogen C. beticola,sugar beets of the tolerant genotype 1K0088 and of the susceptiblegenotype 3S0057 are cultured in addition to transgentic plants undergreenhouse conditions. Two weeks before the intended inoculation 20 V8vegetable juice plates (40% Albani vegetable juice) are inoculated withfour different C. beticola isolates and incubated at 25° C. Immediatelybefore inoculation, the fungus grown agar is homogenized with 0.5 lwater in a high performance agitator (UM5 Universal, Stephan). Theconcentration of mycelium fragments and fungus spores in the homogenateis determined with the help of a counting chamber. The inoculum densityis adjusted to a concentration of 100,000 fragments/ml by dilution withwater. The diluted homogenate is spread onto 12 week old sugar beet withthe help of a backpack sprayer (Gloria 176T). For control purposes,plants are sprayed with fungus-free agar homogenate. The plants areincubated after inoculation for 4 days at 25° C. and 95% humidity in agreenhouse. After day four, humidity is reduced to 60-70%. Fourteen andtwenty-one days after inoculation, the infection of the leaves by fungusand agar inoculated plants are optically determined.

Transgenetic sugar beets show, e.g. in case of the use of promoters,which are active in above-ground organs, an increased resistance towardsthe leaf parasites Cercospora beticola and also an increased resistancetowards Ramularia beticola and Erysiphe betae. Sugar beets, in which thepathogen defense genes are root specifically expressed, comprise anincreased resistance towards the parasites Rhizoctonia solani andAphanomyces cochlioides.

Surprisingly, the organ specific constitutive expression of pathogendefense genes does not lead to low-growing or reduced yield, oftenobserved with the constitutive expression in the entire plant. Anadditional advantage of root and above organ specific gene expression isthat the resistance mediated gene products are only formed in the organto be protected.

Genes, which gene products have an indirect antifungal activity, need aparticularly careful expression control in order to avoid negativeconsequences of an undesired activation of the plant defense. Oneexample for an indirect antifungal activity is the effect, which isderived from the coexpression of a plant resistance (R) gene incombination with an avirulence (avr) gene in a plant cell. Simultaneousexpression of R and avr gene leads to an intensive activation of theplant defense and needs a strict regulation. The regulation is obtainedby either placing the R gene and/or the avr gene under the control ofthe pathogen inducible promoter.

Pathogen inducible promoters, as, e.g. the Vst1 promoter from wine oftenshow in above-ground organs of transgenetic plants in addition to alocal, specific activation after infection with commercially relevantphytopathogenic fungus, an unspecific activation in the root region(Stahl et al., 1995). This promoter activation is induced bypythopathologically harmless microorganisms and renders promoters asVst1 unsuitable for the expression of an R or avr gene. However, by anorgan or tissue specific constitutive expression of the R or avr geneunder the control of promoters C1 and C2, which are specific forabove-ground organs, a pathogen activatible promoter like, e.g. the Vst1promoter can be used for the expression of the corresponding avr or Rgene. Thus, the R/avr gene concept can be realized organ specifically.

In order to obtain the use of the R/avr gene concept in above-groundorgans of sugar beet, potato, brassica and Arabidopsis thaliana, therespective R gene is fused with the promoter C1 and C2, respectively. Inthe case of potato, as an R gene, e.g. the Cf-9 gene of tomato is used,and for brassica and A. thaliana the RPM1 gene from A. thaliana is used.The corresponding avr gene (avr9 from Cladosporium fulvum for potato andavrB from Pseudomonas spp. for brassica and A. thaliana) istranscriptionally or translationally combined with a pathogen sensitivepromoter like Vst1, hcbt2 or with one of the chimeric pathogen sensitivepromoters (PCT/EP99/08710, Chimeric promoters capable of mediated geneexpression in plants upon pathogen infection and uses thereof). Bothexpression cassettes are integrated into a binary vector, like BIN19. ByA. tumefaciens mediated transformation, the C1/C2 promoter-R-genecombinations and the fusion between pathogen sensitive promoter and avrgene are transformed into sugar beet, potato, brassica and Arabidopsisthaliana. Subsequent resistant testing shows that sugar beetstransformed with the R/avr gene combination using the C1 and C2promoters, comprise a very high resistance towards the leaf parasitesCercospora beticola, Ramularia beticola and Erysiphe betae. Resistancetesting of transgenetic potatoes show a very high resistance towardsPhytophthora infestans, and with brassica plants a very high resistancetowards Phoma lingam, Sclerotinia sclerotiorum and Cylindro-sporiumconcentricum is observed. Transgenetic A. thaliana show a resistancetowards Peronospora parasitica. It is a common feature of alltransgenetic plants that they do not show any undesired necrosis ormisformation in the root region, which would indicate an undesiredactivation of R/avr system.

Repression of Pathogen Defense in Non-Infected Tissues by Using RootSpecific Promoters 2-1-48 and 2-1-36 or Promoters C1 and C2, Which areActive in Above-Ground Organs

An activation of pathogen defense in non-infected tissues may havenegative consequences on the plant development and the yield due torelated consequences as cell death and conversion of the cellmetabolism. A trigger for the undesired activation can be the use ofpathogen sensitive promoters with insufficient specificity incombination with pathogen defense genes. An example for this problem isthe use of pathogen sensitive promoters for the expression of anavirulence gene in combination with the corresponding resistance gene.

Root specific promoters can be used in order to suppress an undesiredactivation of, e.g. pathogen sensitive promoters in the root.

Thereby, said promoters can be used for the conversion of the R/avrconcept besides “background activity”.

TABLE 1 Comparison of transcript accumulation of the root specificexpressed ene 2-1-48 in different organs of the sugar beet 4 6 10 12 1622 Organ weeks weeks weeks weeks weeks weeks source leaf 86.4^(1,4) 53.634.9 0.7 86.5 19.1 sink leaf 56.1² n.d.³ 22.1 34.0 30.8 8.2 petiolen.d.³ 74.7 49.4 72.0 36.0 16.5 taproot 2231.0 2506.0 2581.2 6082.81408.5 681.3 lateral root n.d.³ 1633.0 486.8 4932.2 1403.5 833.1 ¹Assource leaf, the first leaf pair was chosen on 4 week old sugar beet.²As sink leaf, the seed leaves were chosen on 4 week old sugar beet.³n.d. = not done. ⁴Measurement values are given in psl (photo stimulatedluminescence) units. Total cell RNA was isolated after sowings atvarious development time points (4, 6, 10, 12, 16, 22 weeks) from sinkand source leaves, from petioles, taproots and lateral roots of sugarbeets and investigated by an RNA blot analysis. As a hybridizationprobe, the cDNA fragment 2-1-48 was used. The transcript amount formedby promoter activity was quantified with the help of a phosphoimager,and is depicted in the table for each investigation time point. Thebackground activity of the nylon filters was once determined for eachfilter and subtracted from the measurement values. The background valuefor the analysis of time points 4-10 weeks is 164.9 psl and is 215.7 pslat time points 12-22 weeks.

TABLE 2 Comparison of transcript accumulation of the root specificexpressed gene 2-1-36 in different organs of the sugar beet 4 6 10 12 1622 Organ weeks weeks weeks weeks weeks weeks source leaf  3.4^(1,4) 2.07.8 0 9.5 5.5 sink leaf  0² 4.8 7.3 3.6 12.4 11 setiole n.d.³ 9.8 4.75.3 15.4 13.7 taproot 67.2 102.7 136.2 803.3 546.4 518.7 lateral rootn.d.³ 71.5 177.4 888.0 363.1 874.0 ¹As source leaf, the first leaf pairwas chosen on 4 week old sugar beet. ²As sink leaf, the seed leaves werechosen on 4 week old sugar beet. ³n.d. = not done. ⁴Measurement valuesare given in psl (photo stimulated luminescence) units. Total cell RNAwas isolated after sowings at various development time points (4, 6, 10,12, 16, 22 weeks) from sink and source leaves, from petioles, taprootsand lateral roots of sugar beets and investigated by an RNA blotanalysis. As a hybridization probe, the cDNA fragment 2-1-36 was used.The transcript amount formed by promoter activity was quantified withthe help of a phosphoimager, and is depicted in the table for eachinvestigation time point. The background activity of the nylon filterswas once determined for each filter and subtracted from the measurementvalues. The background value for the analysis of time points 4-10 weeksis 145 psl and is 212.67 psl at time points 12-22 weeks.

TABLE 3 Comparison of transcript accumulation of gene 2-3-9 in differentorgans of sugar beet 4 6 10 12 16 22 Organ weeks weeks weeks weeks weeksweeks source leaf 3788^(1,4) 2084.2 249.6 1117.9 2073.7 1634.4 sink leaf1927.7² 2239.3 2067.7 3976.2 3471.2 4269.6 petiole n.d.³ 1237.2 9601589.7 1140.6 774.1 taproot   0 14.2 0 17.8 27.3 13.2 lateral root n.d.³0 0 19.4 26.5 0 ¹As source leaf, the first leaf pair was chosen on 4week old sugar beet. ²As sink leaf, the seed leaves were chosen on 4week old sugar beet. ³n.d. = not done. ⁴Measurement values are given inpsl (photo stimulated luminescence) units. Total cell RNA was isolatedafter sowings at various development time points (4, 6, 10, 12, 16, 22weeks) from sink and source leaves, from petioles, taproots and lateralroots of sugar beets and investigated by an RNA blot analysis. As ahybridization probe, the cDNA fragment 2-3-9 was used. The transcriptamount formed by promoter activity was quantified with the help of aphosphoimager, and is depicted in the table for each investigation timepoint. The background activity of the nylon filters was determined onfour independent positions, respectively, and the mean value of thebackground was subtracted from the measurement values. The backgroundvalues for the analysis of the time points 4-10 weeks are 155.1 psl as amean value (individual values: 150.7; 150.1; 141.7 and 177.9), and attime points 12-22 weeks the mean value is 155.9 psl (individual values:149.3; 150.4; 178.3 and 145.8).

TABLE 4 Detection of C1 promoter activity in sugar beet leaves byballistic transformation plate 1 plate 2 plate 3 Photinus/ Photinus/Photinus/ mean Renilla Renilla Renilla plate 1 plate 2 plate 3 valueleaf luciferase luciferase luciferase normal. normal. normal. normal.construct type activity¹ activity¹ activity¹ gene expression² geneexpression² gene expression² gene expression² pc1L- small 8590 629424431 7.8 7.7 8.6 8.0 1097 119120 91725 294751 pc1L- large 16149 80789253 6.0 6.8 16.1 9.6 1097 278403 131141 72079 pc1L- small 4751 48116008 2.5 2.8 4.4 3.2 7126 192866 175901 144327 pc1L- large 13465 85711231 5.7 11.6 3.6 7.0 7126 246870 87835 42334 without small 228 DNA12500 without large 300 DNA 16369 ¹relative light units ²the normalizedexpression values are calculated as follows: (Photinus measuredvalue_((construct)) − Photinus measured value_((without DNA))/Renillameasured value_((construct)) − Renilla measured value_((without DNA)) ×100.

LITERATURE

Altschul, S. F. et al. (1990). Basic Local Alignment search tool, J.Mol. Biol. 215: 403-410An, G. (1987). Binary Ti vectors for plant transformation and promoteranalysis. Methods Enzymol. 153, 292-305.Berghall, S., Briggs, S., Elsegood, S. E., Eronen, L., Kuusisto, J. O.,Philip, E. J., Theobald, T. C., and Walliander, P. (1997). The role ofsugar beet invertase related enzymes during growth, storage andprocessing. Zuckerind. 122, 520-530.Cai, D. et al., (1997). Positional cloning of a gene for nematoderesistance in sugar beet. Science 275, 832-834.DE 4207358 A1 (Institut für Genbiologische Forschung Berlin GmbH).Expressionskassette und Plasmid zur schliesszellenspezifischenExpression und ihre Verwendung zur Her-stellung transgenerPflanzenzellen und Pflanzen.De Greve, H., Dhaese, P., Seurinck, J., Lemmers, M., Van Montagu, M.,and Schell, J. (1982). Nucleotide sequence and transcript map of theAgrobacterium tumefaciens Ti plasmid-encoded Octopine synthase gene. JMol. Appl. Genet. 1 (6), 499-511.Depicker, A., Stachel, S., Dhaese, P. Zambryski, P., and Goodman, H. M.(1982). Nopaline synthase: Transcript mapping and DNA sequence. J Mol.Appl. Genet. 1 (6), 561-573.Diatchenko, L., Lau, Y.-F. C., Campbell, A. P., Chenchik, A., Moqadam,F., Huang, B., Lukyanov, S., Lukyanov, K., Gurskaya, N., Sverdlov, E.D., and Siebert, P. D. (1996). Suppression subtractive hybridization: Amethod for generating differentially regulated or tissue-specific cDNAprobes and libraries. Proc. Natl. Acad. Sci. USA 93, 6025-6030.Dzelzkàlns, V. A., Thorsness, M. K., Dwyer, K. G., Baxter, J. S.,Balent, M. A., Nasrallah, M. E., and Nasrallah, J. B. (1993). Distinctcis-acting elements direct pistil-specific and pollen-specific activityof the Brassica S locus glycoprotein gene promoter. Plant Cell 5,855-863.EP 0344029 B1. (Plant Genetic Systems, N. V. 1040 Brussel). Plants withmodified stamen cells.EP 99/08710, Chimeric promoters capable of mediating gene expression inplants upon pathogen infection and uses thereof.Giuliano, G., Pichersky, E., Malik, V. S., Timko, M. P., Scolnik, P. A.,and Cashmore, A. R. (1988). An evolutionarily conserved protein bindingsequence upstream of a plant light regulated gene. Proc. Natl. Acad.Sci. USA 85, 7089-7093.Greiner, S., Krausgrill, S., and Rausch, T. (1998). Cloning of a tobaccoapoplasmic invertase inhibitor: Proof of function of the recombinantprotein and expression analysis during plant development. Plant Physiol.116, 733-742.Hesse, H., and Willmitzer, L. (1996). Expression analysis of a sucrosesynthase gene from sugar beet (Beta vulgaris L). Plant Mol Biol 30,863-872.Höfgen R. & Hesse H.: DE 19607697, granted Sep. 4, 1998/WO 97/32027,published Apr. 9, 1997Horsch, R. B., Fry, J. E., Hoffmann, N. L., Rogers, S. G., Fraley, R. T.(1985). A simple and general method for transferring genes into plants.Science 227, 1229-1231.Klein. K. (1995). Die Bedeutung von Saccharose-Stoffwechselgenen für diebakterielle Isomaltulose-Herstellung. Dissertation UniversistätStuttgart.Lam, E. and Chua, N. H. (1989). ASF-2: A factor that binds to thecauliflower mosaic virus 35S promoter and a conserved GATA motif in cabpromoters. Plant Cell 1, 1147-1156.Logemann, J., Schell, J., and Willmitzer, L. (1987). Improved method forthe isolation of RNA from plant tissue. Anal. Biochem. 163, 16-20.Odell, J. T. Nagy, F., and Chua, N.-H. (1985). Identification of DNAsequences required for activity of the cauliflower mosaic virus 35Spromoter. Nature 313, 810-812.Piechulla, B., Merforth, N., and Rudolph, B. (1999). Identification oftomato Lhc promoter regions necessary for circadian expression. PlantMol. Biol. 38, 655-662.Saghai-Maroof, M. A., Solimanm, K. M., Jorgensen, R. A., ans Allard, R.W. (1984). Ribosomal DNA spacer length polymorphism in barley: mendelianinheritance, chromosomal location and population dynamics. Proc. Natl.Acad. Sci. USA 81, 8014-8018.

Sambrook , J., Fritsch, E. F., and Maniatis, T (1989). In MolecularCloning, A Laboratory Manual. (Cold Spring Harbor Laboratory Press, NewYork).

Stahl, D. J., Fischer, R., Dettendorfer, J., Sauerbrey, E., Hain, R.,and Nehls, R. (1995). Molecular analysis of the pathogen inducedexpression of the resveratrol synthase gene in transgenic plants. 4^(th)International Workshop on Pathogenesis related proteins in plants:Biology and biotechnological potential. Kloster Irsee, September 3-7.U.S. Pat. No. 5,608,150A (Monsanto Company). Fruit specific promoters.Van der Vossen, E. A. G., Rouppe van der Voort, J. N. A. M., Kanyuka,K., Bendahmane, A., Sandbrink, H., Baulcombe, D. C., Bakker, J.,Stiekema, W. J., and Klein-Lankhorst, R. M. (2000). Homologues of asingle resistance-gene cluster in potato confer resistance to distinctpathogens: a virus and a nematode. Plant Journal 23(5), 567-576.Velten, J., Velten, L., Hain, R., and Schell, J. (1984). Isolation of adual promoter fragment from Ti plasmid of Agrobacterium tumefaciens.EMBO J. 12, 2723-2730.WO 94/02619 (Pioneer Hi-Breed International, Inc.) A brassicaeregulatory sequence for root-specific or root abundant gene expression.WO 97/28268 (The Minister of Agriculture and Agri-Food Canada). Promoterfrom tobacco.WO 97/27307 (Agritope, Inc). Raspberry promoters for expression oftransgenes in plants.WO 97/32027 (Max-Planck-Gesellschaft zur Förderung der Wissenschaften).Sugarbeet storage-root-tissue-specific regulon.WO 98/18940 (BASF Aktiengesellschaft). Leaf-specific gene expression intransgenic plants.WO/98/45460 (Rhone-Poulenc Agro). A sunflower albumin 5′regulatoryregion for the modification of plant seed lipid composition.Zhou, D. X. (1999). Regulatory mechanism of plant gene transcription byGT-elements and GT-factors. Trends in Plant Sciences 4, 210-214.

1. An isolated root-specific promoter comprising a nucleic acid sequence selected from the group consisting of: a. SEQ ID NO: 2; b. the polynucleotide sequence complementary to the full length of SEQ ID NO: 2; c. a sequence having root-specific promoter activity that hybridizes under stringent hybridizing conditions to SEQ ID NO: 2; and d. a sequence that hybridizes under stringent hybridizing conditions to a sequence complementary to the full length of SEQ ID NO: 2, wherein said sequence that hybridizes has root-specific promoter activity; wherein the stringent hybridizing conditions are either: (1) hybridizing in 4×SSC at 65° C. and subsequent multiple washing in 0.1×SSC at 65° C. for a total of 1 hour; or (2) hybridizing at 68° C. in 0.25 M sodium phosphate, pH 7.2, 7% SDS, 1 mM EDTA and 1% BSA for 16 hours and subsequent washing twice with 2×SSC and 0.1% SDS at 68° C.
 2. The isolated promoter of claim 1 inserted into a vector or operably connected to a mobile genetic element.
 3. A transgenic eukaryotic or a transgenic prokaryotic host cell comprising a root-specific promoter that is operably linked to a transgene, wherein said promoter comprises a nucleotide sequence selected from the group consisting of: a. SEQ ID NO: 2 having a promoter activity; b. the polynucleotide sequence complementary to the full length of SEQ ID NO: 2; c. a sequence having root-specific promoter activity that hybridizes under stringent hybridizing conditions to SEQ ID NO: 2; and d. a sequence that hybridizes under stringent hybridizing conditions to a sequence complementary to the full length of SEQ ID NO: 2, wherein said sequence that hybridizes has root-specific promoter activity; wherein the stringent hybridizing conditions are either: (1) hybridizing in 4×SSC at 65° C. and subsequent multiple washing in 0.1×SSC at 65° C. for a total of 1 hour; or (2) hybridizing at 68° C. in 0.25 M sodium phosphate, pH 7.2, 7% SDS, 1 mM EDTA and 1% BSA for 16 hours and subsequent washing twice with 2×SSC and 0.1% SDS at 68° C.
 4. A transgenic plant comprising a root-specific promoter that is operably linked to a transgene, wherein said promoter comprises a nucleotide sequence selected from the group consisting of: a. SEQ ID NO: 2 having a promoter activity; b. a sequence complementary to the full length of SEQ ID NO: 2; c. the polynucleotide sequence having root-specific promoter activity that hybridizes under stringent hybridizing conditions to SEQ ID NO: 2; and d. a sequence that hybridizes under stringent hybridizing conditions to a sequence complementary to the full length of SEQ ID NO: 2, wherein said sequence that hybridizes has root-specific promoter activity; wherein the stringent hybridizing conditions are either: (1) hybridizing in 4×SSC at 65° C. and subsequent multiple washing in 0.1×SSC at 65° C. for a total of 1 hour; or (2) hybridizing at 68° C. in 0.25 M sodium phosphate, pH 7.2, 7% SDS, 1 mM EDTA and 1% BSA for 16 hours and subsequent washing twice with 2×SSC and 0.1% SDS at 68° C.
 5. The transgenic plant according to claim 4, wherein said transgenic plant is Beta vulgaris, and wherein said promoter is active in the root, but not the above ground organs, of Beta vulgaris.
 6. The transgenic plant according to claim 4, wherein said transgenic plant is further characterized by the expression of a transgene exclusively in a below-ground organ, wherein said expression is obtainable by transformation of a plant cell with said promoter that is operably linked to a transgene.
 7. A transgenic seed produced by said transgenic plant of claim
 4. 8. The transgenic plant according to claim 6, wherein said below-ground organ is a root.
 9. The transgenic plant according to claim 6, wherein said plant belongs to species Beta vulgaris, and wherein said promoter is active in the root, but not the above ground organs, of Beta vulgaris.
 10. The transgenic plant according to claim 6, wherein said transgenic plant is characterized by: a. amending a carbohydrate metabolism; b. avoiding a loss of a storage substance; c. expressing an invertase inhibitor; d. expressing a fructosyl transferase; e. expressing a levan sucrose; f. expressing a gene coding for a transporter protein for a N-compound; or g. developing a feature that increases at least one of resistance and tolerance towards pathogens.
 11. A method of: a. amending carbohydrate metabolism, b. avoiding loss of storage substance, c. increasing at least one of resistance and tolerance towards a pathogen, or d. expressing from a transgene: an invertase inhibitor, a fructosyl transferase, a levan sucrose, or a transporter protein for a N-compound, in a transgenic plant, said method comprising: (1) transforming a plant cell with a root-specific promoter, wherein said promoter is operably linked to said transgene, and (2) subsequently regenerating a transgenic plant from said plant cell, wherein said promoter comprises a nucleotide sequence selected from the group consisting of: i. SEQ ID NO: 2; ii. the polynucleotide sequence complementary to the full length of SEQ ID NO: 2; iii. a sequence having root-specific promoter activity that hybridizes under stringent hybridizing conditions to SEQ ID NO: 2; and iv. a sequence that hybridizes under stringent hybridizing conditions to a sequence complementary to the full length of SEQ ID NO: 2 wherein said sequence that hybridizes has root-specific promoter activity; wherein the stringent hybridizing conditions are either: (A) hybridizing in 4×SSC at 65° C. and subsequent multiple washing in 0.1×SSC at 65° C. for a total of 1 hour; or (B) hybridizing at 68° C. in 0.25 M sodium phosphate, pH 7.2, 7% SDS, 1 mM EDTA and 1% BSA for 16 hours and subsequent washing twice with 2×SSC and 0.1% SDS at 68° C.
 12. An isolated promoter comprising a nucleotide sequence selected from the group consisting of: a. SEQ ID NO: 2; and b. the polynucleotide sequence complementary to the full length of SEQ ID NO:
 2. 13. The isolated promoter of claim 12 inserted into a vector or operably connected to a mobile genetic element.
 14. A transgenic eukaryotic or a transgenic prokaryotic host cell comprising a promoter that is operably linked to a transgene, wherein said promoter comprises a nucleotide sequence selected from the group consisting of: a. SEQ ID NO: 2 having a promoter activity; and b. the polynucleotide sequence complementary to the full length of SEQ ID NO:
 2. 15. A transgenic plant comprising a promoter that is operably linked to a transgene, wherein said promoter comprises a nucleotide sequence selected from the group consisting of: a. SEQ ID NO: 2 having a promoter activity; and b. the polynucleotide sequence complementary to the full length of SEQ ID NO:
 2. 16. The transgenic plant according to claim 15 wherein said transgenic plant is Beta vulgaris, and wherein said promoter is active in the root, but not the above-ground organs, of Beta vulgaris.
 17. The transgenic plant according to claim 15, wherein said transgenic plant is further characterized by the expression of a transgene exclusively in a below-ground organ, wherein said expression is obtainable by transformation of a plant cell with said promoter that is operably linked to a transgene.
 18. A transgenic seed produced by said transgenic plant of claim
 15. 19. The transgenic plant according to claim 17, wherein said below-ground organ is a root.
 20. The transgenic plant according to claim 17, wherein said plant belongs to species Beta vulgaris, and wherein said promoter is active in the root, but not the above-ground organs, of Beta vulgaris.
 21. The transgenic plant according to claim 17, wherein said transgenic plant is characterized by: a. amending a carbohydrate metabolism; b. avoiding a loss of a storage substance; c. expressing an invertase inhibitor; d. expressing a fructosyl transferase; e. expressing a levan sucrose; f. expressing a gene coding for a transporter protein for an N-compound; or g. developing a feature that increases at least one of resistance and tolerance towards pathogens.
 22. A method of: a. amending carbohydrate metabolism; b. avoiding loss of storage substance; c. increasing at least one of resistance and tolerance towards a pathogen; or d. expressing from a transgene: an invertase inhibitor, a fructosyl transferase, a levan sucrose, or a transporter protein from an N-compound, in a transgenic plant, said method comprising: (1) transforming a plant cell with a promoter, wherein said promoter is operably linked to said transgene; and (2) subsequently regenerating a transgenic plant from said plant cell, wherein said promoter comprises a nucleotide sequence selected from the group consisting of: i. SEQ ID NO: 2; and ii. the polynucleotide sequence complementary to SEQ ID NO:
 2. 